Pitfalls in Performing and Interpreting Inferior Petrosal Sinus Sampling: Personal Experience and Literature Review.

CONTEXT: Inferior petrosal sinus sampling (IPSS) helps differentiate the source of ACTH-dependent hypercortisolism in patients with inconclusive biochemical testing and imaging, and is considered the gold standard for distinguishing Cushing disease (CD) from ectopic ACTH syndrome. We present a comprehensive approach to interpreting IPSS results by examining several real cases. EVIDENCE ACQUISITION: We performed a comprehensive review of the IPSS literature using PubMed since IPSS was first described in 1977. EVIDENCE SYNTHESIS: IPSS cannot be used to confirm the diagnosis of ACTH-dependent Cushing syndrome (CS). It is essential to establish ACTH-dependent hypercortisolism before the procedure. IPSS must be performed by an experienced interventional or neuroradiologist because successful sinus cannulation relies on operator experience. In patients with suspected cyclical CS, it is important to demonstrate the presence of hypercortisolism before IPSS. Concurrent measurement of IPS prolactin levels is useful to confirm adequate IPS venous efflux. This is essential in patients who lack an IPS-to-peripheral (IPS:P) ACTH gradient, suggesting an ectopic source. The prolactin-adjusted IPS:P ACTH ratio can improve differentiation between CD and ectopic ACTH syndrome when there is a lack of proper IPS venous efflux. In patients who have unilateral successful IPS cannulation, a contralateral source cannot be excluded. The value of the intersinus ACTH ratio to predict tumor lateralization may be improved using a prolactin-adjusted ACTH ratio, but this requires further evaluation. CONCLUSION: A stepwise approach in performing and interpreting IPSS will provide clinicians with the best information from this important but delicate procedure.

Prolactin measurement during inferior petrosal sinus sampling improves the localization of pituitary adenomas in Cushing's disease.

OBJECTIVE: Inferior petrosal sinus sampling (IPSS) distinguishes pituitary-dependent Cushing's disease (CD) from ectopic ACTH syndrome with a high degree of certainty, but has not been reliable in predicting the location of an adenoma within the pituitary gland. We investigated whether prolactin measurements during IPSS would improve pituitary tumour localization. METHODS: Fifty-four patients with suspected ACTH-dependent Cushing's syndrome who underwent IPSS between 1997 and 2009 were studied retrospectively. Twenty-eight patients who had an identifiable tumour that stained for ACTH on histopathology are the subject of this study. Intersinus ACTH gradients before and after adjustment for prolactin were compared with surgical findings and pathology. RESULTS: Magnetic resonance imaging localized a pituitary adenoma in 17/28 (61%) patients. Using a maximum intersinus ACTH gradient of ≥1·4 before or after CRH stimulation, we could diagnose the tumour location correctly in 15/28 (54%) patients. By comparison, tumour lateralization by means of a dominant (≥1·4) prolactin-adjusted ACTH intersinus gradient was correct in 21/28 (75%) patients (P = 0·041). Tumour localization was correct in 23/28 (82%) patients when MRI and prolactin-adjusted ratio data were combined. Fourteen patients with proper bilateral IPS venous sampling (as determined by concurrent IPS to peripheral prolactin ratio ≥1·8) either had correct localization of the tumour (n = 12) or had a central lesion (n = 2). In none of these 14 patients was the dominant prolactin-adjusted ACTH ratio associated with a tumour on the opposite side of the gland. CONCLUSION: Prolactin measurement, during IPSS, improves our ability to correctly localize the pituitary adenoma site in CD.

Reduction of false-negative results in inferior petrosal sinus sampling with simultaneous prolactin and corticotropin measurement.

OBJECTIVE: To investigate the value of prolactin as an independent marker of catheter placement to improve the diagnostic accuracy of inferior petrosal sinus sampling (IPSS) in patients with corticotropin-dependent Cushing syndrome. METHODS: In this retrospective cohort study, we reviewed hospital records of patients who underwent IPSS procedures at the Cleveland Clinic between 1997 and 2009. Serum prolactin and plasma corticotropin levels were measured prospectively in peripheral and inferior petrosal sinus (IPS) samples. RESULTS: Forty-one patients underwent 42 IPSS procedures at our institution during the study period. Among 35 patients with Cushing disease, 1 patient had erroneous IPSS results: all pre-corticotropin-releasing hormone (CRH) and post-CRH IPS to peripheral (IPS:P) ACTH ratios were less than 2 and less than 3, respectively. Despite radiologic evidence of appropriate IPS catheter placement, concurrent IPS:P prolactin ratios indicated that successful IPS venous sampling was not achieved. A second case with equivocal IPSS results could also be explained by corresponding IPS:P prolactin ratios. During IPSS, all patients with an identifiable ACTH-staining adenoma localizing to 1 side of the pituitary gland (n = 22) who demonstrated absent IPS:P ACTH gradients (<2 before or <3 after CRH administration) on the ipsilateral side of the corticotroph adenoma had corresponding IPS:P prolactin ratios less than 1.3. CONCLUSIONS: Measurement of prolactin during IPSS testing may reduce false-negative results in patients with Cushing disease who do not demonstrate an appropriate central-to-peripheral ACTH gradient. In our series, all false-negative IPS:P ACTH ratios had a corresponding IPS:P prolactin ratio less than 1.3.

Taking vitamin D with the largest meal improves absorption and results in higher serum levels of 25-hydroxyvitamin D.

Many patients treated for vitamin D deficiency fail to achieve an adequate serum level of 25-hydroxyvitamin D [25(OH)D] despite high doses of ergo- or cholecalciferol. The objective of this study was to determine whether administration of vitamin D supplement with the largest meal of the day would improve absorption and increase serum levels of 25(OH)D. This was a prospective cohort study in an ambulatory tertiary-care referral center. Patients seen at the Cleveland Clinic Foundation Bone Clinic for the treatment of vitamin D deficiency who were not responding to treatment make up the study group. Subjects were instructed to take their usual vitamin D supplement with the largest meal of the day. The main outcome measure was the serum 259(OH)D level after 2 to 3 months. Seventeen patients were analyzed. The mean age (+/-SD) and sex (F/M) ratio were 64.5 +/- 11.0 years and 13 females and 4 males, respectively. The dose of 25(OH)D ranged from 1000 to 50,000 IU daily. The mean baseline serum 25(OH)D level (+/-SD) was 30.5 +/- 4.7 ng/mL (range 21.6 to 38.8 ng/mL). The mean serum 25(OH)D level after diet modification (+/-SD) was 47.2 +/- 10.9 ng/mL (range 34.7 to 74.0 ng/mL, p < .01). Overall, the average serum 25(OH)D level increased by 56.7% +/- 36.7%. A subgroup analysis based on the weekly dose of vitamin D was performed, and a similar trend was observed.Thus it is concluded that taking vitamin D with the largest meal improves absorption and results in about a 50% increase in serum levels of 25(OH)D levels achieved. Similar increases were observed in a wide range of vitamin D doses taken for a variety of medical conditions.

A patient with pseudo-Addison's disease and falsely elevated thyroxine due to interference in serum cortisol and free thyroxine immunoassays by two different mechanisms.

Interference in immunoassays is a widely recognized problem, which could potentially lead to unnecessary investigations and treatment. We describe a case where interference in a cortisol immunoassay led to a falsely low serum cortisol concentration and interference in the free thyroxine assay led to falsely elevated serum thyroxine concentrations, in the same patient. A 42-year-old woman with documented hypothyroidism underwent a synacthen test for suspected adrenal insufficiency. Previous thyroid function tests had been discordant and difficult to interpret, with elevated thyroxine and non-suppressed thyroid-stimulating hormone. The synacthen test showed a subnormal cortisol response and she was commenced on cortisol replacement. Clinically, it was doubted whether she had true adrenal insufficiency and it was thought that the cortisol results might be artefactually low due to assay interference. Cortisol was measured by an alternative immunoassay, before and after incubation in an antibody blocking tube ('Scantibodies'), after heat treatment and also after treatment with Protein A. The results supported assay interference and cortisol 'replacement' was stopped. Thyroxine had been discontinued although thyroid function tests (TFTs) were significantly different between analytical platforms, also consistent with interference. Thyroxine replacement was restarted and once on a stable dose, the discrepancy in TFTs was also investigated by similar procedures as for cortisol. Good clinician-laboratory interface and laboratory work-up of patients with results that are discordant from the clinical findings can reduce unnecessary investigation and inappropriate treatment.

Transition state stabilization by six arginines clustered in the active site of creatine kinase.

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.

Study of hematological and biochemical parameters in runners completing a standard marathon.

OBJECTIVE: To study hematological and biochemical parameters prospectively in runners completing a standard 42.2-km marathon run. To determine the incidence of hyponatremia in runners, and whether consumption of nonsteroidal anti-inflammatory medications (NSAIDs) was associated with alterations in serum biochemical parameters. DESIGN: Observational cohort study. SETTING: City of Christchurch (New Zealand) Marathon, June 2002. PARTICIPANTS: One hundred fifty-five of the 296 athletes entered in the 2002 City of Christchurch Marathon were enrolled in the study. MAIN OUTCOME MEASURES: Athletes were weighed at race registration and immediately after the race. Blood was drawn postrace for measurement of serum sodium, potassium, creatinine, and urea concentrations and for hematological analysis (hemoglobin concentration, hematocrit, leukocyte distribution). RESULTS: Complete data sets including prerace and postrace weights, and postrace hematological and biochemical analyses were collected on 134 marathon finishers. Postrace serum sodium concentrations were directly related to changes in body weight (P < 0.0001). There were no cases of biochemical or symptomatic hyponatremia. Thirteen percent of runners had taken an NSAID in the 24 hours prior to the race. Mean values for serum creatinine (P = 0.03) and serum potassium (P = 0.007) concentrations were significantly higher in runners who had taken an NSAID. No athlete who had taken an NSAID had a postrace serum creatinine concentration less than 0.09 mmol/L. Ninety-eight percent of runners had a postrace leukocytosis (mean white cell count, 18.97 b/L), of which the major component was a raised neutrophil count (mean neutrophil count, 15.69 b/L). CONCLUSIONS: This study found no cases of hyponatremia in runners completing a standard distance marathon. This finding relates to a marathon run under ideal conditions (minimal climatic stress) and in which there were fewer aid stations (every 5 km) than is common in North American marathons (every 1.6 km). Also, aggressive hydration practices were not promoted. Consumption of NSAIDs in the 24 hours prior to distance running was associated with altered renal function.

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase.

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.